Prostate-specific membrane antigen (PSMA) is a clinically validated target in metastatic castration-resistant prostate cancer (mCRPC) that is being prosecuted by a number of different modalities in the clinic. CD3 T-cell engagers (TCEs) targeting PSMA have shown promise in preclinical and early clinical studies, but generating a molecule with a therapeutic window that enables efficacious dosing in patients has been a barrier to development. Here, we present preclinical in vitro and in vivo data on novel PSMA x CD3 TCEs developed using our TCE platform. To address the challenges of TCE development for mCRPC, we screened and identified hundreds of diverse PSMA- and CD3-binding antibodies with different affinities, epitopes, and biophysical properties using our proprietary antibody screening platform. From there, we engineered large panels of OrthomabTM PSMA x CD3 bispecifics, varying TCE parameters that impact function. Detailed in vitro functional assessment and biophysical characterization assays were conducted to identify antibodies with promising functional and developability profiles. IgG-like bispecifics comprised of PSMA- and CD3-binding arms with finely tuned affinity for each target were generated. PSMA binding epitopes were assessed using cryo-electron microscopy, and TCE function was measured using in vitro T cell co-culture assays. TCEs binding membrane-proximal epitopes drove optimal immune synapse formation, leading to potent killing of cells expressing high (C4-2) and low (22Rv1) levels of PSMA with EC50 values in the picomolar range. Molecules show target-dependent T-cell activation with no killing of a low PSMA-expressing cell line (DU-145) in vitro. Further, select molecules show robust CD4+/CD8+ T-cell activation and proliferation in the presence of target cells, as well as sustained killing of target cells over time in a repeat challenge assay. Finally, molecules evaluated in vivo in a humanized C4-2 xenograft mouse model demonstrated anti-tumor activity and a favorable IgG-like pharmacokinetic profile. In summary, we engineered and assessed hundreds of PSMA x CD3 TCEs at high-throughput, conducted detailed in vitro functional and biophysical characterization, and identified molecules with promising preclinical in vivo efficacy that supports further evaluation and development towards the clinic. Peter Bergqvist, Alaa Amash, Kelly Bullock, Lauren Clifford, Patrick Farber, Jessica Fernandes Scortecci, Ingrid Knarston, Tallie Kuang, Ahn Lee, Amy Lee, Cindy-Lee Crichlow, Franco Li, Matt Mai, Stephanie K. Masterman, Janice Reimer, Eduardo Solano Salgado, Raffi Tonikian, Christopher Williamson, Allison Goodman, Lindsay DeVorkin. PSMA x CD3 T-cell engagers show preclinical efficacy for the treatment of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6012.

Abstract Accessing peptides from intracellular tumor antigens displayed on MHC class I (pMHC) can expand the T-cell engager (TCE) target pool for solid tumor applications. A challenge limiting TCE development for pMHC targets is the identification of rare, high-affinity, and high-specificity pMHC-binders. In this work, we describe TCEs in a 1 × 1 format with potent tumor-cell killing activity and high specificity for MAGE-A4, a pMHC tumor antigen expressed in multiple cancer types. We identified functional MAGE-A4 x CD3 TCEs with high specificity and affinity to a human MAGE-A4 peptide sequence of 10 amino acids presented on MHC-I (HLA-A:02*01). We strategically selected MAGE-A4-binders with high binding specificity for pMAGE-A4230-239, but not MHC-I or many closely related MHC-restricted peptides. We then paired them with diverse and developable CD3-binders with a range of binding affinities, subunit specificities, and binding kinetic profiles. High-throughput in vitro functional characterization of hundreds of these TCEs enabled identification of molecules with optimal T-cell dependent cellular cytotoxicity in MAGE-A4-expressing tumor cell lines, with comparable tumor-killing activity and cytokine release to a clinical-stage TCE in a 2 × 1 format. Previous studies have highlighted unique challenges associated with pMHC targets, including their potential for cross-reactive binding to pMHCs on healthy tissues, and the absence of pharmacologically relevant species for preclinical toxicity testing. To address these challenges, we have designed and implemented an in vitro and in silico workflow to identify antibody binding to potential off-target peptides. Using this approach, we selected TCEs with several pMHC binding orientations and high specificity and affinity. These TCEs killed tumor cell lines endogenously expressing MAGE-A4 pMHCs and not isogenic cells with MAGE-A4 knocked out. Results demonstrate that our high-resolution, multiparametric antibody discovery capabilities can identify pMHC-binding antibodies with desired functional and specificity attributes. This approach unlocks the discovery and development of optimal TCEs against complex pMHC antigens. Citation Format: Davide Tortora, Peter Bergqvist, Allison Goodman, Ryan Blackler, Nathalie Blamey, Stefania Carrara, Lauren Chong, Gabrielle Conaghan, Cindy-Lee Crichlow, Valentine de Puyraimond, Harveer Dhupar, Patrick Farber, Jessica Fernandes Scortecci, Kate Gibson, Rodrigo Goya, Ahn Lee, Franco Li, Tova Pinsky, Craig Robb, Patrick Rowe, Antonios Samiotakis, Eduardo Solano Salgado, Ping Xiang, Irene Yu, Kelly Bullock, Tara Fernandez, Stephanie K. Masterman, Kush Dalal, Tim Jacobs, Bryan C. Barnhart. Functional and specific T-cell engagers against a peptide-MHC tumor target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2373.

Abstract In this study, we used a functional approach to generate CD3 T-cell engager (TCE) molecules targeting prostate-specific membrane antigen (PSMA). We present data on the robust in vitro characterization of T-cell activity and selection of molecules for further assessment. TCE function is dictated by the interplay between multiple factors that form the immune synapse, including binding kinetics, binding geometry, and epitopes of the CD3- and tumor-binding arms. To diversify immune synapse parameters, we engineer hundreds of bispecific molecules from highly diverse parental antibodies, and employ a high-throughput process to select molecules with desired functional properties. We applied this approach to engineer CD3 TCEs targeting PSMA. PSMA is an attractive target for the treatment of castration-resistant prostate cancer due to its high expression in prostate cancer cells and low relative expression in other tissues. We discovered hundreds of diverse, fully human PSMA-binding antibodies using high-throughput single B-cell screening and selected human/cynomolgus cross-reactive binders with a broad range of affinities and epitopes. We paired these with human/cynomolgus cross-reactive CD3-binding antibodies from our TCE platform to generate 180 bispecific PSMA x CD3 TCEs. CD3-binding antibodies covered several orders of magnitude of affinity (nanomolar to micromolar) and included binders specific for different CD3 subunits. We used high-throughput T-cell dependent cellular cytotoxicity (TDCC) and cytokine release assays to identify molecules with desired functional profiles. Bispecifics selected for further assessment had functional profiles spanning a range of properties that have been observed in clinical molecules, including potent killing and low cytokine release. To profile T-cell properties that are associated with anti-tumor immune responses, we subjected selected molecules to a battery of additional in vitro functional assessments, including proliferation, cytokine and chemokine production, and TDCC using cynomolgus T cells. Results demonstrate that a functional approach that begins with highly diverse CD3- and tumor-binding antibodies can generate promising TCEs with minimal need for protein engineering and optimization. Citation Format: Valentine de Puyraimond, Matt Mai, Alaa Amash, Nathalie Blamey, Gabrielle Conaghan, Jessica Fernandes Scortecci, Allison Goodman, Ahn Lee, Irene Yu, Franziska von Bank, Kate Caldwell, Lauren Clifford, Ingrid Knarston, Kelly Bullock, Melissa Cid, Cindy-Lee Crichlow, Lindsay Devorkin, Fiona Dickson, Patrick Farber, Stefan Hannie, Courteney Lai, Vivian Li, Stephanie K. Masterman, Iwona Niemietz, Philippe Pouliot, Ping Xiang, Bryan C. Barnhart, Raffi Tonikian. Development of PSMA x CD3 T-cell engagers using an integrated, functional approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6359.

Abstract In this study, we present data from four T-cell engager (TCE) programs in which we leveraged our highly diverse CD3-binding antibodies to generate functional bispecifics for each tumor target. By aggregating tumor-cell killing and cytokine data across four tumor targets with different properties and target expression levels, we have gained novel insights into target-specific considerations for the development of CD3 TCEs. TCEs recapitulate a synapse between T cells and target cells. T cell function downstream of this synapse is determined by the complex interplay between multiple independent factors. These include properties of the CD3- and tumor-binding arms, such as binding kinetics and epitope, as well as target-dependent factors such as cell type and target density. We used our diverse CD3 antibodies to engineer bispecific TCEs against four solid tumor targets: PSMA, B7-H4, 5T4, and the peptide-MHC target MAGE-A4. For each target, we engineered and characterized hundreds of bispecific molecules using high-throughput assays, including T-cell dependent tumor-cell killing, cytokine release, developability assessments, and structural studies. Here, we compare data from across these programs to elucidate the impact of target-dependent parameters on TCE function. These data provide important insights for the selection of appropriate TCE targets and efficient design of bispecific antibodies with high therapeutic potential. Citation Format: Matt Mai, Raffi Tonikian, Peter Bergqvist, Alaa Amash, Nathalie Blamey, Gabrielle Conaghan, Valentine de Puyraimond, Patrick Farber, Allison Goodman, Ahn Lee, Jessica Fernandes Scortecci, Cindy-Lee Crichlow, Akram Khodabandehloo, Tova Pinsky, Kate Caldwell, Jessica Patterson, Philippe Pouliot, Davide Tortora, Oscar Urtatiz, Ping Xiang, Irene Yu, Kirstin Brown, Kelly Bullock, Andrea Chee, Stephanie K. Masterman, Neil Aubuchon, Lindsay Devorkin, Bryan C. Barnhart, Tim Jacobs. Target-dependent considerations for the design of bispecific T-cell engagers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1868.

Bispecific antibodies (bsAb) and multispecific antibodies (msAb) encompass a diverse variety of formats that can concurrently bind multiple epitopes, unlocking mechanisms to address previously difficult-to-treat or incurable diseases. Early assessment of candidate developability enables demotion of antibodies with low potential and promotion of the most promising candidates for further development. Protein-based therapies have a stringent set of developability requirements in order to be competitive (e.g. high-concentration formulation, and long half-life) and their assessment requires a robust toolkit of methods, few of which are validated for interrogating bsAbs/msAbs. Important considerations when assessing the developability of bsAbs/msAbs include their molecular format, likelihood for immunogenicity, specificity, stability, and potential for high-volume production. Here, we summarize the critical aspects of developability assessment, and provide guidance on how to develop a comprehensive plan tailored to a given bsAb/msAb.

Background Effective CD3 T-cell engagers (TCEs) must balance potency with potential toxicities, such as cytokine release syndrome. The CD3- and tumor-binding arms should function together to maximize tumor-cell killing while fine-tuning cytokine release. However, most TCEs in clinical development are derived from a small number of CD3-binding antibodies, such as SP34–2, limiting the pairs of parental antibodies that allow for optimal immunological synapse formation. To address these barriers, AbCellera developed a complete TCE platform: a discovery engine to identify diverse antibodies with high specificity for tumor targets, a portfolio of fully-human, developable, CD3-binding antibodies that are distinct from SP34–2, a clinically-validated multispecific engineering platform, and a high-throughput strategy to rapidly identify TCE candidates. Previously, we demonstrated that identical CD3-binding antibodies result in variable functional profiles when paired with different tumor targets, supporting the value of beginning with diverse parental antibodies. But the full extent to which CD3-binding parameters impact TCE function has not been well-understood due to the limited number of anti-CD3 antibodies that have been used to generate TCEs. In this study, we designed and engineered our largest panel of bispecific TCEs to date and used high-throughput functional assessments to gain novel insights into how CD3-binding properties impact function. We present here an overview of those insights, along with a demonstration of how we leverage these lessons to rationally select CD3-binders for different tumor targets. Methods To deepen our understanding of CD3-binding parameters that impact function, we paired hundreds of CD3-binders with a single tumor-binding paratope. We assessed bispecifics using our high-throughput primary T cell functional assays, including tumor-cell killing and release of IFNγ, TNFα, IL-2, and IL-6. We performed a clustering analysis to identify distinct functional profiles, and aggregated these data with extensive analyses of our CD3-binders to gain insights into how CD3-binding properties impact function. Results We compared TCE functional readouts across a range of monovalent CD3-binding parameters, including affinity, on- and off-rates, subunit specificity, and epitopes, revealing the impact of several parameters on TCE function. We then leveraged these insights, combined with our functional clustering analysis, to select diverse CD3-binding antibodies for multiple bispecific TCE programs. Conclusions We integrated large parental and bispecific antibody datasets to gain insights into parameters that impact TCE function. By combining these insights with our previous data demonstrating the impact of tumor target on TCE function, we have developed a strategy to rationally select tumor- and CD3-binding antibody pairs for each tumor target.

Background Bispecific T-cell engagers (TCEs) activate the immune system to Figureht cancer. TCEs that target peptides displayed on major histocompatibility complexes (pMHC) have shown promise for unlocking previously inaccessible intracellular tumor-specific antigens. Bispecific antibodies have the potential to overcome challenges associated with other pMHC-targeting modalities, such as soluble T cell receptors, by eliminating the need for extensive engineering of endogenous TCRs to produce molecules with the requisite affinity and specificity.1 Peptides derived from melanoma-associated antigen 4 (MAGE-A4) are presented by MHC class I (MHC-I) in many solid tumors, but rarely in healthy tissues. However, developing TCE therapies against MAGE-A4 pMHCs presents complex and unique challenges. The tumor-binding arm must bind specifically to small (~10 amino acid) MAGE-A4 peptides that are highly homologous to other proteins in the MAGE family, avoid substantial interactions with the MHC complex, bind tumor cells expressing very low levels of the target, and work in concert with the CD3-binding arm to activate T cells. To address these challenges, we have developed a technology platform to discover optimal TCEs that combines hundreds of diverse, fully human CD3-binding antibodies with antibody discovery, engineering, functional screening, and development capabilities. We discovered diverse and developable human antibodies that showed high specificity and affinity to a human MAGE-A4 peptide presented on MHC-I (HLA:02*01).We strategically selected and paired a panel of these MAGE-A4-pMHC-binding antibodies with diverse CD3-binders and used our high-throughput characterization platform to identify TCEs with optimal specificity and functional profiles. Methods We used our OrthoMab™ multispecifics platform and high-throughput expression to generate more than 400 MAGE-A4-pMHCxCD3 TCEs in bispecific and multispecific formats, and measured purity by mass spectrometry, aSEC, and CE-SDS. We assessed the functional activity of these TCEs in high-throughput T cell cytotoxicity and cytokine release assays. We investigated potential off-target cross-reactivity in MAGE-A4-pMHC-binding parental antibodies with a rationally designed screening library based on X-scan binding data. In-depth binding structural and kinetic assessments were also performed to map antibody-epitope interactions. Results We identified a panel of MAGE-A4-pMHC-specific TCEs that induce T-cell dependent cellular cytotoxicity while maintaining low cytokine release in MAGE-A4-positive tumor cell lines. We mapped multiple binding parameters including target specificity, kinetics, and affinity to the functional properties of TCEs in different structural formats to identify candidates for further development. Conclusions We have identified a panel of functional and specific MAGE-A4-pMHCxCD3 TCEs as potential immunotherapies against solid tumors. Reference Holland CJ, Crean RM, Pentier JM, et al. Specificity of bispecific T cell receptors and antibodies targeting peptide-HLA. J Clin Invest. 2020;130(5):2673–2688.

Functional biologics design is a multi-objective optimization problem often with competing design objectives. We report on a novel deep learning based protein sequence prediction framework, ZymeSwapNet, that can be customized to handle a wide range of quantifiable design objectives, a current limitation of traditional protein design methods. We train a simple convolutional neural network (1D-CNN) on nonredundant curated protein crystal structures, using a set of geometric and topological features that describes a local protein environment, to predict the likelihood of each amino acid type for residue sites in the design region. While the model can be directly used to rank templates derived from mutagenesis campaigns, we extend the scope by developing a sequence/mutation generator that optimizes the desired multivariate distribution using a Monte-Carlo sampling. Using a case study – the design of a stable heterodimeric Fc (HetFc) antibody domain – we show that we can further include a Metropolis criterion to bias the sampling to enhance features such as the heterodimeric binding specificity, in addition to original sampling objective of enhancing stability. We demonstrate that ZymeSwapNet can generate stable HetFc designs, within minutes that had taken several rounds of rational structure and physical force-field based modeling attempts.

In order to gain further insight into the molecular mechanism of arginine-dependent operator recognition by the hexameric Escherichia coli arginine repressor we have probed protein-DNA interactions in vitro and in vivo. We have extensively applied the chemical modification-protection and premodification-interference approach to two operators, the natural operator overlapping the P2 promoter of the carAB operon and a fully symmetrical consensus sequence. Backbone contacts were revealed by hydroxyl radical footprinting and phosphate ethylation interference. Base-specific contacts to purines and pyrimidines were revealed by methylation protection and premodification interference, KMnO4 and NH2OH.HCl-specific modification of thymine and cytosine residues, base-removal (depurination and depyrimidation), and base substitution (uracil and inosine). Additional information on the groove specificity of repressor binding was obtained by small ligand binding interference (distamycin and methyl green). In vivo, we measured the effects on the repressibility of 24 single base-pair substitutions obtained by saturation mutagenesis of half an Arg box in the carAB operator. The results of these experiments point to the conclusion that a hexameric arginine repressor molecule covers four turns of the helix, makes base-specific contacts to at least one guanine (G4 or G4′) and two thymine (T3, T13′, or T3′, T13) residues in each one of four consecutive major grooves on one face of the helix and with four A-T/T-A base-pairs, comprising the adenine residues A9, 9′, 12, 12′ and the thymine residues T10, 10′, 11, 11′, in the two outermost minor grooves of the operator, on the very same face of the DNA molecule. The hydrophobic 5-methyl groups of four thymine residues (T3, 3′, 13, 13′) in each Arg box contribute to major groove-specific recognition via hydrophobic and/or van der Waals interactions. The importance of minor groove contacts was further supported by the drastic effect of distamycin binding interference. In vivo, the most pronounced drops in repressibility were occasioned by mutations at positions 10 (A → G or C), 11 (T → A or G) and 12 (A → G, T or C).

Protein phase separation is implicated in formation of membraneless organelles, signaling puncta and the nuclear pore. Multivalent interactions of modular binding domains and their target motifs can drive phase separation. However, forces promoting the more common phase separation of intrinsically disordered regions are less understood, with suggested roles for multivalent cation-pi, pi-pi, and charge interactions and the hydrophobic effect. Known phase-separating proteins are enriched in pi-orbital containing residues and thus we analyzed pi-interactions in folded proteins. We found that pi-pi interactions involving non-aromatic groups are widespread, underestimated by force-fields used in structure calculations and correlated with solvation and lack of regular secondary structure, properties associated with disordered regions. We present a phase separation predictive algorithm based on pi interaction frequency, highlighting proteins involved in biomaterials and RNA processing.

Significance The cell is divided into compartments where specific biochemical functions are performed. These compartments can be delineated by membranes or through phase separation of proteins or protein and nucleic acids to form membraneless organelles. The latter situation occurs with an intrinsically disordered region of Ddx4, a major constituent of germ granules. The nature of the interior of membraneless organelles is poorly understood. Here, we use NMR to show that the intrinsically disordered Ddx4 region remains disordered and highly dynamic in the phase-separated state, while diffusing as slowly as a particle the size of a bacterial cell. Ddx4 molecules form a network of interactions on phase separation, providing an alternative environment to that found in membrane-encapsulated organelles.

Amyloids are fibrillar protein superstructures that are commonly associated with diseases in humans and with physiological functions in various organisms. The precise mechanisms of amyloid formation remain to be elucidated. Surprisingly, we discovered that a bacterial Escherichia coli chaperone-like ATPase, regulatory ATPase variant A (RavA), and specifically the LARA domain in RavA, forms amyloids under acidic conditions at elevated temperatures. RavA is involved in modulating the proper assembly of membrane respiratory complexes. LARA contains an N-terminal loop region followed by a β-sandwich-like folded core. Several approaches, including nuclear magnetic resonance spectroscopy and molecular dynamics simulations, were used to determine the mechanism by which LARA switches to an amyloid state. These studies revealed that the folded core of LARA is amyloidogenic and is protected by its N-terminal loop. At low pH and high temperatures, the interaction of the N-terminal loop with the folded core is disrupted, leading to amyloid formation.

Allosteric transmission of information between distant sites in biological macromolecules often involves collective transitions between active and inactive conformations. Nuclear magnetic resonance (NMR) spectroscopy can yield detailed information on these dynamics. In particular, relaxation dispersion techniques provide structural, dynamic, and mechanistic information on conformational transitions occurring on the millisecond to microsecond timescales. In this review, we provide an overview of the theory and analysis of Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR experiments and briefly describe their application to the study of allosteric dynamics in the homeodomain from the PBX transcription factor (PBX-HD). CPMG NMR data show that local folding (helix/coil) transitions in one part of PBX-HD help to communicate information between two distant binding sites. Furthermore, the combination of CPMG and other spin relaxation data show that this region can also undergo local misfolding, reminiscent of conformational ensemble models of allostery.

ClpB/Hsp100 is an ATP-dependent disaggregase that solubilizes and reactivates protein aggregates in cooperation with the DnaK/Hsp70 chaperone system. The ClpB-substrate interaction is mediated by conserved tyrosine residues located in flexible loops in nucleotide-binding domain-1 that extend into the ClpB central pore. In addition to the tyrosines, the ClpB N-terminal domain (NTD) was suggested to provide a second substrate-binding site; however, the manner in which the NTD recognizes and binds substrate proteins has remained elusive. Herein, we present an NMR spectroscopy study to structurally characterize the NTD-substrate interaction. We show that the NTD includes a substrate-binding groove that specifically recognizes exposed hydrophobic stretches in unfolded or aggregated client proteins. Using an optimized segmental labeling technique in combination with methyl-transverse relaxation optimized spectroscopy (TROSY) NMR, the interaction of client proteins with both the NTD and the pore-loop tyrosines in the 580-kDa ClpB hexamer has been characterized. Unlike contacts with the tyrosines, the NTD-substrate interaction is independent of the ClpB nucleotide state and protein conformational changes that result from ATP hydrolysis. The NTD interaction destabilizes client proteins, priming them for subsequent unfolding and translocation. Mutations in the NTD substrate-binding groove are shown to have a dramatic effect on protein translocation through the ClpB central pore, suggesting that, before their interaction with substrates, the NTDs block the translocation channel. Together, our findings provide both a detailed characterization of the NTD-substrate complex and insight into the functional regulatory role of the ClpB NTD in protein disaggregation.

Deletion of Phe-508 (F508del) in the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) results in destabilization of the domain, intramolecular interactions involving the domain, and the entire channel. The destabilization caused by F508del manifests itself in defective channel processing and channel gating defects. Here, we present NMR studies of the effect of F508del and the I539T stabilizing mutation on NBD1 dynamics, with a view to understanding these changes in stability. Qualitatively, F508del NMR spectra exhibit significantly more peak broadening than WT spectra due to the enhanced intermediate time scale (millisecond to microsecond) motions in the mutant. Unexpectedly, studies of fast (nanosecond to picosecond) motions revealed that F508del NBD1 tumbles more rapidly in solution than WT NBD1. Whereas F508del tumbles at a rate nearly consistent with the monomeric state, the WT protein tumbles significantly more slowly. Paramagnetic relaxation enhancement experiments confirm that NBD1 homodimerizes in solution in the expected head-to-tail orientation. NMR spectra of WT NBD1 reveal significant concentration-dependent chemical shift perturbations consistent with NBD1 dimerization. Chemical shift analysis suggests that the more rapid tumbling of F508del is the result of an impaired ability to dimerize. Based on previously published crystal structures and NMR spectra of various NBD1 mutants, we propose that deletion of Phe-508 affects Q-loop conformational sampling in a manner that inhibits dimerization. These results provide a potential mechanism for inhibition of channel opening by F508del and support the dimer interface as a target for cystic fibrosis therapeutics.

Cells chemically isolate molecules in compartments to both facilitate and regulate their interactions. In addition to membrane-encapsulated compartments, cells can form proteinaceous and membraneless organelles, including nucleoli, Cajal and PML bodies, and stress granules. The principles that determine when and why these structures form have remained elusive. Here, we demonstrate that the disordered tails of Ddx4, a primary constituent of nuage or germ granules, form phase-separated organelles both in live cells and in vitro. These bodies are stabilized by patterned electrostatic interactions that are highly sensitive to temperature, ionic strength, arginine methylation, and splicing. Sequence determinants are used to identify proteins found in both membraneless organelles and cell adhesion. Moreover, the bodies provide an alternative solvent environment that can concentrate single-stranded DNA but largely exclude double-stranded DNA. We propose that phase separation of disordered proteins containing weakly interacting blocks is a general mechanism for forming regulated, membraneless organelles.

Intrinsically disordered proteins and regions (IDPs/IDRs), which do not have stable secondary and tertiary structure, are capable of adopting different structural states. Many IDPs/IDRs populate conformationally heterogeneous monomeric states or engage in discrete interactions with other proteins, leading to folding upon binding or retaining significant disorder in the bound state. Others are involved in large-scale association having different degrees of order, from more defined fibers, to variably networked gels and to disordered liquid demixed states or droplets. These latter have been suggested to provide the physical basis for cellular membrane-less organelles such as the nucleolus. We have studied the N-terminal disordered region of Ddx4, an RNA DEAD-box helicase that is essential for formation of a class of membrane-less organelles termed nuage or germ granules functioning in spermatogenesis. When expressed in HeLa cells, the protein forms spherical, micron-sized, liquid-like cellular organelles. In vitro, it phase separates to form droplets with similar morphological and dynamic properties to the organelles observed in cells. Phase separation is sensitive to salt, pointing to the importance of electrostatic interactions. The sequence features of the disordered N-terminus of Ddx4 that underlie phase separation include clustering of charged residues into blocks of net positive and negative charge, with over-representation of FG/GF pairs and RG/GR pairs within the positive blocks. Perturbations of these properties disrupt phase separation, pointing to multi-valent cation-pi interactions playing an important role. The transient sampling of multi-valent interactions in self-association of Ddx4 extends previous observations of dynamic multi-valent interactions in discrete complexes of IDPs/IDRs, such as for Sic1:Cdc4 binding. The insights obtained from these and ongoing biophysical studies of Ddx4 will be valuable for developing a general understanding of the biogenesis and disassembly of membrane-less cellular organelles.

The yeast cyclin-dependent kinase inhibitor Sic1 is a disordered protein that, upon multisite phosphorylation, forms a dynamic complex with the Cdc4 subunit of an SCF ubiquitin ligase. To understand the multisite phosphorylation dependence of the Sic1:Cdc4 interaction, which ultimately leads to a sharp cell cycle transition, the conformational properties of the disordered Sic1 N-terminal targeting region were studied using single-molecule fluorescence spectroscopy. Multiple conformational populations with different sensitivities to charge screening were identified by performing experiments in nondenaturing salts and ionic denaturants. Both the end-to-end distance and the hydrodynamic radius decrease monotonically with increasing the salt concentration, and a rollover of the chain dimensions in high denaturant conditions is observed. The data were fit to the polyelectrolyte binding-screening model, yielding parameters such as the excluded volume of the uncharged chain and the binding constant to denaturant. An overall scaling factor of ∼1.2 was needed for fitting the data, which implies that Sic1 cannot be approximated by a random Gaussian chain. Fluorescence correlation spectroscopy reveals Sic1 structure fluctuations occurring on both fast (10-100 ns) and slow (∼10 ms) time scales, with the fast phase absent in low salt solutions. The results of this study provide direct evidence that long-range intrachain electrostatic repulsions are a significant factor for the conformational landscape of Sic1, and support the role of electrostatics in determining the overall shape and hydrodynamic properties of intrinsically disordered proteins.